Both efficiency and safety of gene delivery vectors must be considered from the early stages of development to ensure successful introduction of human gene therapy into clinical practice. The objective of this research proposal, which is a key step in our long-term goal to develop vectors for use in human cancer gene therapy, is to design an improved, more efficient and less toxic means of delivering nucleic acids to target cells using linear polyethylenimines sensitive to redox-potential gradients (r/PEI). It is hypothesized that introduction of disulfide bonds into the backbone of linear PEI will improve overall efficiency of gene delivery process due to glutathione (GSH)-dependent intracellular reduction of the disulfide bonds, which will reduce cytotoxicity and enhance intracellular disassembly rates of r/PEI-containing vectors. The central hypothesis will be tested and overall objectives of this application accomplished by pursuing three specific aims. First, it will be determined if cytotoxicity of r/PEI is inversely correlated with rates of intracellular degradation by altering concentrations of intracellular GSH. Second, the working hypothesis will be tested that increased rates of disassembly of r/PEI complexes promote better transcription and translation of the delivered DMA and mRNA. Using two Bcl2 overexpressing human cancer cell lines, it will be tested whether the enhanced concentration and redistribution of GSH into the nucleus associated with Bcl2 overexpression permits achieving enhanced selectivity of transfection. Third, it will be investigated whether in vitro cytotoxicity and transfection activity of r/PEI vectors correlate with their in vivo properties by evaluating their hepatotoxicity and their ability to mediate targeted delivery to model tumors. Overall, the results obtained in this proposal are significant because they are expected to provide new insights into the gene delivery process by synthetic vectors and enable improved rational design of vectors with enhanced safety and intracellular disassembly controlled by physiological redox-potential gradients. As such, these studies are expected to establish that degradation of r/PEI by intracellular GSH not only reduces cytotoxicity but also permits controlling intracellular disassembly of the vectors, thus opening the possibility to use high molecular weight PEI for efficient delivery of both therapeutic DNA and mRNA. It is also expected that enhanced selectivity of DMA and mRNA transfection will be achieved in Bcl2-overexpressing tumors.